In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step: strand exchange between homologous parental DNAs. In vitro, the product of this strand exchange reaction is a joint molecule composed a single-strand DNA joined to one end of a linear duplex DNA. We have established a new paradigm for this homologous pairing. In essence, that recombinases such as the E. coli RecA protein can form a novel DNA triplex (R-form DNA) in which the third strand may have any arbitrary sequence and must have a parallel orientation with respect to the phosphodiester backbone of the identical strand in the duplex. In order to understand the mechanism and structures involved in greater detail we have endeavored to miniaturize the reaction. In the past, we have shown that short oligonucleotides can be used as the substrates. Recently we have determined that a 20 amino acid peptide that includes loop L2 of RecA can promote the key reaction of the whole RecA protein: pairing (targeting) of a single stranded DNA to its homologous site on a duplex DNA. In the course of the reactions the peptide binds to both substrate DNAs, unstacks the single-stranded DNA, and assumes a b structure. This b-structure can be induced by environmental conditions that facilitate intermolecular interactions present when they are bound to DNA, such as high pH and high peptide concentration. The DNA-pairing domain of RecA visualized by EM self-assembles into a filamentous structure like RecA. It is possible that the two DNAs align and pair on an extended b-sheet of L2s in the whole RecA. We have recently extended the range of functions shared between the whole RecA protein and the DNA pairing peptides by showing that these miniRecA peptides promote not only pairing of homologous DNAs but also homology-dependent strand exchange between three colinear oligonucleotide strands. In order to understand the function of L2 we have generated by site-directed mutagenesis all possible mutants of residues 193-212 in the whole RecA protein (380 mutants). The in vivo phenotype of these mutants with respect to recombination and UV and mitomycin resistance was determined. An analysis of these results suggested that L2 may be involved in most aspects of RecA function. For example, as RecA is an ATP-dependent DNA binding protein and a DNA-dependent ATPase, we asked whether the loop might be directly involved in these allosteric interactions. We have been able to show that ATP, but not ADP, interacts with the arginine (Arg196) within L2 peptides and that this interaction induces the active b-structure conformation of the peptides. Experiments with mutant RecA proteins indicate that Arg196 binds to both DNA and the g-phosphate of ATP and is essential for the cooperativity between DNA and ATP binding. We suggest a mechanism for ATP hydrolysis by RecA that is similar to those proposed for heterotrimeric G proteins. We are studying the function of other domains of RecA and how they interact with L2 by selecting for second-site revertants of L2 mutants.